Microarrays: an introduction and their applications to plant and medical research

Microarrays an introduction and their applications to plant and medical research Microarray an introduction and their applications to plant and medical research Nelzo C Ereful A presentation on Microa[.]

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Microarray: an introduction and their

applications to plant and medical

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Nelzo Ereful Crop Bioinformatics

Outline

1 “Omics” organization overview

3 Steps in Microarray analysis

4 Applications of microarray

2 A partial view of transcriptomics

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Omics organization: an overview

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Another way of looking at it

Taken from Goodacre (2005)

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Transcriptomics

global analysis of gene expression

produced by the genome at any one time

cell population, often using high-throughput

techniques such DNA microarray technology.

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Transcriptomics

Analysis techniques

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What is microarray?

• Typically a glass slide where DNA molecules

are fixed

• Allows simultaneous measurement the level

of expression of thousands of genes

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Landscape

subarray

Slide

gene spot

pingroup

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Affymetrix GeneChip

Oligonucleotide:

•11-25 mer (short oligo)

•50-70 mer (long oligo)

http://en.wikipedia.org/wiki/Image:Affymetrix-microarray.jpg

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Applications of microarray

Drug Discovery and development

Evolutionary and ecological genomics

classification

Gene expression (stress)

Microarray

SNP

detection

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Pre-microarray analysis questions

NO YES

at a particular condition or time?

(Gene expression analysis)

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and technical replicates

Design the series

of hybridization

Experimental

design

Technical performance

Obtain the samples Isolate total RNA Label cDNA or mRNA

Perform the hybridizations

Scan the slides

or chips

Statistical analysis

Extract fluorescence intensities

Normalize data to remove biases t-tests for pairwise comparisons

ANOVA for multifactorial designs

Data mining

Cluster analysis and pattern recognition

Study lists of genome ontologies

Design validation and follow-up experiments Search for regulatory motifs

Gibson, G and SV Muse, 2004

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Another way of looking at it

Joseph Clarke and Tong Zhu, 2006

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Experimental design

contrast each experimental sample against a common reference sample

ex: cancer type 1, type 2, etc vs

normal person

(modeled after agricultural field trials) useful when

an experiment already involves multiple factors ex: variety and stress

(with dye swap) for three or more

conditions

ex: well-watered, control and no water (rice)

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Reference sample design

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CONSULT YOUR

STATISTICIAN!

Experimental design

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Experimental design

Drought stress

(Katherisan, 2005)

Define objective:

Assess drought-induced expression

among three varieties (IR64, Apo,

Azucena)

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Sample questions

What particular tissue?

What stage of the plant?

What variety (ies)?

What statistical design?

Sampling time?

What conditions?

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1 2 3 5 6 7 8 9 12 18 20 days

Fl Not watered

Field experimental procedure

(Katherisan, 2005)

Apo, IR64

Azucena

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mRNA extraction

Images by Microarray Mediabook http://gcat.davidson.edu/Pirelli/index.htm

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cDNA synthesis and fluorescent dye labeling

1 cDNA Synthesis

2 Dyes are

incorporated

Cy3 (green)

Control/ well-watered Cy5 (red) Experimental/ stressed

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cDNA are mixed

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Construction of cDNA library

(Katherisan, 2005)

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Microarray analysis

Hypothetical genome profile

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Slides printing using GeneTAC printer

Microtiter plates Glass slides

3000 spots per slide

PCR products

from >9000 genes

Images by Dr John Bennett, IRRI

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Hybridize overnight

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GeneTAC Hyb station

Automated and manual hybridization chambers

Manual hyb chamber

in water bath

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The hybridization process

Quackenbush, 2006

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Image processing by Laser scanning

Microarray Mediabook on scanning

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Images captured by scanner

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Slides Scanning

10K rice panicle cDNA library printed at IRRI

59 K oligo array from BGI, Beijing

22K chips from Agilent

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Data analysis

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Expression ratio

R G

T=

Reference/ Control Test/ Experimental

18000

11600 13000 15500 5600

1200 600

800 2500

6500 10900

13500 16500

17500

22.5 6.2

2.0 1.0

0.4 0.07

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Fold change

18000

11600 13000 15500 5600

1200 600

800 2500

6500 10900

13500 16500

17500

22.5 6.2

2.0 1.0

0.4 0.07

0.03

R G

2.5 14.3

33.3

Reciprocal

Fold Induction Fold Repression

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Asymmetrical distribution

22.5 6.2

2.0 1.0

0.4 0.07

0.03

Image by Microarray Mediabook http://gcat.davidson.edu/Pirelli/index.htm

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• log 2 transform

Transformation

22.5 6.2

2.0 1.0

0.4 0.07

0.03 -5.1 -3.8 -1.3 0 1.0 2.6 4.5

Induction Repression

Image by Microarray Mediabook http://gcat.davidson.edu/Pirelli/index.htm

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• Differences in the amount of mRNA samples

• Spatial positions of the spots

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Reporting your results

Microarray

Gene

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Transformed colors

Blue-yellow

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Dendogram

Higher correlation between genes

Lower correlation between genes

0.8 0.85 0.9

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Results

Another slide

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Medical application of microarray analysis

treatment of cancer may be

tailored to the needs of that

individual’s gene expression

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Tumor classification

Quackenbush 2006; Retrieved from http://content.nejm.org/cgi/content/full/354/23/2463

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Expression matrix

Quackenbush 2006; Retrieved from

http://content.nejm.org/cgi/content/full/354/23/2463

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Cluster Analysis

Quackenbush 2006; Retrieved from

http://content.nejm.org/cgi/content/full/354/23/2463

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References

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Microarray and asthma

Something more

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tolerant ATGTGTGGAGGAGAAGTGATCCCCGCCGACATGCCGGCGGCGCCGTTCACGCCACGCCAC 60 susceptible -ATGCCGGCGGCGCCGTTCACGCCACGCCAC 30

******************************

tolerant GGCGACGGCGAGACATGGGTTGACAGAAAGAGGAGGAACAAGAAGAAGAGGAAGCGCGGC 120 susceptible GGCGACGGCGAGACATGGGTTGACAGAAAGAGGAGGAACAAGAAGAAGAGGAAGCGCGGC 90

************************************************************

tolerant GCCGACGAAGAATGGGAGGCCGCCTTCCAGGAGTTCATGGCTGCTGACGACGACGACGAC 180 susceptible GCCGACGAAGAATGGGAGGCCGCCTTCCAGGAGTTCATGGCTGCTGACGACGACGACGAC 150 ************************************************************

tolerant GGCGGCGGACTCGTGTTAAGTAGTAAATCTTTGGTGTTGAGGTCACCAGGTGAAAATGAT 240 susceptible GGCGGCGGACTCGTGTTAAGTAGTAAATCTTTGGTGTTGAGGTCACCAGGTGAAAATGAT 210 ************************************************************

tolerant GCAGGCCGGGGCGCCGCCGCCACCATGTCCATGCCGCTGGACCCCGTGACCGAGGAGGCC 300 susceptible GCAGGCCGGGGCGCCGCCGCCACCATGTCCATGCCGCTGGACCCCGTGACCGAGGAGGCC 270 ************************************************************

tolerant GAGCCGGCGGTGGCTGAGAAGCCTCGCCGGCGCCGGCCGAGGCGGAGCTACGAGTACCAC 360 susceptible GAGCCGGCGGTGGCTGAGAAGCCTCGCCGGCGCCGGCCGAGGCGGAGCTACGAGTACCAC 330 ************************************************************

tolerant GGCATCCGGCAGCGGCCGTGGGGGCGGTGGTCGTCGGAGATCCGCGACCCCGTCAAGGGC 420 susceptible GGCATCCGGCAGCGGCCGTGGGGGCGGTGGTCGTCGGAGATCCGCGACCCCGTCAAGGGC 390 ************************************************************

tolerant GTCCGCCTCTGGCTCGGCACCTTCGACACCGCCGTCGAAGCCGCGCTCGCCTACGACGCC 480 susceptible GTCCGCCTCTGGCTCGGCACCTTCGACACCGCCGTCGAAGCCGCGCTCGCCTACGACGCC 450 ************************************************************

tolerant GAGGCCCGCCGCATCCACGGCTGGAAAGCCCGGACAAACTTCCCACCCGCCGATCTTTCT 540 susceptible GAGGCCCGCCGCATCCACGGCTGGAAAGCCCGGACAAACTTCCCACCCGCCGATCTTTCT 510 ************************************************************

tolerant TCGCCGCCGCCGCCGTCGCAGCCGCTCTGCTTCTTGCTCAACGACAACGGCCTCATCACA 600 susceptible TCGCCGCCGCCGCCGCCGCAGCCGCTCTGCTTCTTGCTCAACGACAACGGCCTCATCACA 570 *************** ********************************************

tolerant ATCGGAGAAGCGCCGACCGACGACGCCGCGTCGACGTCGACGTCGACGACGGAGGCGTCC 660 susceptible ATCGGAGAAGCGCCGACCGACGACGCCGCGTCGACGTCGACGTCGACGACGGAGGCGTCC 630 ************************************************************

tolerant GGCGACGCGCGCATACAACTGGAGTGCTGCTCGGACGACGTGATGGACAGCCTCCTCGCC 720 susceptible GGCGACGCGCGCATACAGCTGGAGTGCTGCTCGGACGACGTGATGGACAGCCTCCTCGCC 690 ***************** ******************************************

tolerant GGCTACGACGTGGCCAGCGGCGACGACATATGGACATGGACATCTGGAGCCTCCTCCACC 780 susceptible GGCTACGACGTGGCCAGCGGCGACGACATATGGACATGGACATCTGGAGCCTCCTCCACC 750 ************************************************************

tolerant TCTGTTAACCAAGAGATCAAGACCCCATCGATCCACCAAAACATATCATATGCAGGTGCC 840 susceptible TCTGTTAACCAAGAGATCAAGACCCCATCGATCCACCAAAACATATCATATGCAGGTGCC 810 ************************************************************

tolerant CGCCCCATGACTTGTCACTTTAAGAATCATAAAAACACTTTTGTACAAATGGAGTGCTCA 900 susceptible CGCCCCATGACTTGTCACTTTAAGAATCATAAAAACACTTTTGTACAAATGGAGTGCTCA 870 ************************************************************

tolerant ACCATGCTAAACTTACTCAAAGGCCACAAACAATAA 936 susceptible ACCATGCTAAACTTACTCAAAGGCCACAAACAATAA 906

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Tiêu đề	Microarrays: An Introduction and Their Applications to Plant and Medical Research
Tác giả	Nelzo C. Ereful
Trường học	Crop Bioinformatics
Chuyên ngành	Plant and Medical Research
Thể loại	presentation

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Số trang	53
Dung lượng	9,34 MB